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lentivirus infection


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#1 Apple1984

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Posted 22 January 2013 - 01:02 PM

Hi,
I am wondering if anybody has the same experience as me.
I generated lentivirus with a MSCV vector with GFP at the N terminal, encoding full length of my protein.
With the viral stocks, I infected multiple cancer cell lines. i could see green fluorescence under microscope and sorted the cells by flow cytometry. I did see lots of fluorescence after sorting. But the expression levels of the protein didn't change at all, tested by western blot.

I don't understand. If it is because of the cell toxicity of my protein.....? But why i could see so much fluorescence?
I am considering to switch another vector. But i still don't understand what happened to my protein. How could the GFP and my protein be separated?

If anybody can help me figure this out?

Many thanks.

#2 pcrman

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Posted 26 January 2013 - 08:27 PM

What is between the two protein sequences: IRES or 2A peptide? Because the GFP is in front of your gene, it is not surprising that GFP has higher expression than your protein. You may need to consider a different vector.

#3 Apple1984

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Posted 27 January 2013 - 12:45 PM

Hi pcrman,
In the vector, my gene is actually in front of GFP, another word, GFP is at the N-terminal of my gene.




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